Uncontrolled cell proliferation is the insignia of cancer. Cell proliferation in response to various stimuli is manifested by a deregulation of the cell division cycle, the process by which cells multiply and divide. Tumor cells typically have damage to the genes that directly or indirectly regulate progression through the cell division cycle.
Hyperproliferative disease states, including cancer, are characterized by cells rampantly winding through the cell cycle with uncontrolled vigor due to, for example, damage to the genes that directly or indirectly regulate progression through the cycle. Thus, agents that modulate the cell cycle, and thus hyperproliferation, could be used to treat various disease states associated with uncontrolled or unwanted cell proliferation. In addition to cancer chemotherapeutic agents, cell cycle inhibitors are also proposed as antiparasitics (See, Gray et al., Curr. Med. Chem. 6, 859–875 (1999)) and recently demonstrated as potential antivirals (See, Schang et al., J. Virol. 74, 2107–2120 (2000)). Moreover, the applicability of antiproliferative agents may be expanded to treating cardiovascular maladies such as artherosclerosis or restenosis (See Braun-Dullaeus et al., Circulation, 98, 82–89 (1998)), and states of inflammation, such as arthritis (See, Taniguchi et al., Nature Med., 5, 760–767(1999)) or psoriasis.
Mechanisms of cell proliferation are under active investigation at cellular and molecular levels. At the cellular level, de-regulation of signaling pathways, loss of cell cycle controls, unbridled angiogenesis or stimulation of inflammatory pathways are under scrutiny, while at the molecular level, these processes are modulated by various proteins, among which protein kinases are prominent suspects. Overall abatement of proliferation may also result from programmed cell death, or apoptosis, which is also regulated via multiple pathways, some involving proteolytic enzyme proteins.
Among the candidate regulatory proteins, protein kinases are a family of enzymes that catalyze phosphorylation of the hydroxyl group of specific tyrosine, serine, or threonine residues in proteins. Typically, such phosphorylation dramatically perturbs the function of the protein, and thus protein kinases are pivotal in the regulation of a wide variety of cellular processes, including metabolisim, cell proliferation, cell differentiation, and cell survival. Of the many different cellular functions in which the activity of protein kinases is known to be required, some processes represent attractive targets for therapeutic intervention for certain disease states. Two examples are cell-cycle control and angiogenesis, in which protein kinases play a pivotal role; these processes are essential for the growth of solid tumors as well as for other diseases.
CDKs constitute a class of enzymes that play critical roles in regulating the transitions between different phases of the cell cycle, such as the progression from a quiescent stage in G1 (the gap between mitosis and the onset of DNA replication for a new round of cell division) to S (the period of active DNA synthesis), or the progression from G2 to M phase, in which active mitosis and cell-division occur. See, e.g., the articles compiled in Science, vol. 274 (1996), pp. 1643–1677; and Ann. Rev. Cell Dev. Biol., vol. 13 (1997), pp. 261–291. CDK complexes are formed through association of a regulatory cyclin subunit (e.g., cyclin A, B1, B2, D1, D2, D3, and E) and a catalytic kinase subunit (e.g., cdc2 (CDK1), CDK2, CDK4, CDK5, and CDK6). As the name implies, the CDKs display an absolute dependence on the cyclin subunit in order to phosphorylate their target substrates, and different kinase/cyclin pairs function to regulate progression through specific portions of the cell cycle.
The D cyclins are sensitive to extracellular growth signals and become activated in response to mitogens during the G1 phase of the cell cycle. CDK4/cyclin D plays an important role in cell cycle progression by phosphorylating, and thereby inactivating, the retinoblastoma protein (Rb). Hypophosphorylated Rb binds to a family of transcriptional regulators, but upon hyperphosphorylation of Rb by CDK4/cyclin D, these transcription factors are released to activate genes whose products are responsible for S phase progression. Rb phosphorylation and inactivation by CDK4/cyclin D permit passage of the cell beyond the restriction point of the G1 phase, whereupon sensitivity to extracellular growth or inhibitory signals is lost and the cell is committed to cell division. During late G1, Rb is also phosphorylated and inactivated by CDK2/cyclin E, and recent evidence indicates that CDK2/cyclin E can also regulate progression into S phase through a parallel pathway that is independent of Rb phosphorylation (see Lukas et al., “Cyclin E-induced S Phase Without Activation of the pRb/E2F Pathway,” Genes and Dev., vol. 11 (1997), pp. 1479–1492).
The progression from G1 to S phase, accomplished by the action of CDK4/cyclin D and CDK2/cyclin E, is subject to a variety of growth regulatory mechanisms, both negative and positive. Growth stimuli, such as mitogens, cause increased synthesis of cyclin D1 and thus increased functional CDK4. By contrast, cell growth can be “reined in,” in response to DNA damage or negative growth stimuli, by the induction of endogenous inhibitory proteins. These naturally occurring protein inhibitors include p21WAF1/CIP1, p27KIP1, and the p16INK4 family, the latter of which inhibit CDK4 exclusively (see Harper, “Cyclin Dependent Kinase Inhibitors,” Cancer Surv., vol. 29 (1997), pp. 91–107). Aberrations in this control system, particularly those that affect the function of CDK4 and CDK2, are implicated in the advancement of cells to the highly proliferative state characteristic of malignancies, such as familial melanomas, esophageal carcinomas, and pancreatic cancers (see, e.g., Hall and Peters, “Genetic Alterations of Cyclins, Cyclin-Dependent Kinases, and CDK Inhibitors in Human Cancer,” Adv. Cancer Res., vol. 68 (1996), pp. 67–108; and Kamb et al., “A Cell Cycle Regulator Potentially Involved in Genesis of Many Tumor Types,” Science, vol. 264 (1994), pp. 436–440). Over-expression of cyclin D1 is linked to esophageal, breast, and squamous cell carcinomas (see, e.g., DelSal et al., “Cell Cycle and Cancer: Critical Events at the G1 Restriction Point,” Critical Rev. Oncogenesis, vol. 71 (1996), pp. 127–142). Genes encoding the CDK4-specific inhibitors of the p16 family frequently have deletions and mutations in familial melanoma, gliomas, leukemias, sarcomas, and pancreatic, non-small cell lung, and head and neck carcinomas (see Nobori et al., “Deletions of the Cyclin-Dependent Kinase-4 Inhibitor Gene in Multiple Human Cancers,” Nature, vol. 368 (1994), pp. 753–756). Amplification and/or overexpression of cyclin E has also been observed in a wide variety of solid tumors, and elevated cyclin E levels have been correlated with poor prognosis. In addition, the cellular levels of the CDK inhibitor p27, which acts as both a substrate and inhibitor of CDK2/cyclin E, are abnormally low in breast, colon, and prostate cancers, and the expression levels of p27 are inversely correlated with the stage of disease (see Loda et al., “Increased Proteasome-dependent Degradation of the Cyclin-Dependent Kinase Inhibitor p27 in Aggressive Colorectal Carcinomas,” Nature Medicine, vol. 3 (1997), pp. 231–234). Recently there is evidence that CDK4/cyclin D might sequester p27, as reviewed in Sherr, et al., Genes Dev., Vol. 13 (1999), pp. 1501–1512. The p21 proteins also appear to transmit the p53 tumor-suppression signal to the CDKs; thus, the mutation of p53 in approximately 50% of all human cancers may indirectly result in deregulation of CDK activity.
The emerging data provide strong validation for the use of compounds inhibiting CDKs, and CDK4 and CDK2 in particular, as anti-proliferative therapeutic agents. Certain biomolecules have been proposed for this purpose. For example, U.S. Pat. No. 5,621,082 to Xiong et al. discloses nucleic acid encoding of inhibitors of CDK6, and WO 99/06540 for CDK's. Peptides and peptidomimetic inhibitors are described in European Patent Publication No. 0 666 270 A2, Bandara, et al., Nature Biotechnology, Vol. 15 (1997), pp. 896–901 and Chen, et al., Proceedings of the National Academy of Science, USA, Vol. 96 (1999), pp. 4325–4329. Peptide aptamers were identified from screening in Cohen, et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 95 (1998), pp. 14272–14277. Several small molecules have been identified as CDK inhibitors (for recent reviews, see Webster, “The Therapeutic Potential of Targeting the Cell Cycle,” Exp. Opin. Invest. Drugs, vol. 7 (1998), pp. 865–887, and Stover, et al., “Recent advances in protein kinase inhibition: current molecular scaffolds used for inhibitor synthesis,” Current Opinion in Drug Discovery and Development, Vol. 2 (1999), pp. 274–285). The flavone flavopiridol displays modest selectivity for inhibition of CDKs over other kinases, but inhibits CDK4, CDK2, and CDK1 equipotently, with IC50s in the 0.1–0.3 μM range. Flavopiridol is currently in Phase II clinical trials as an oncology chemotherapeutic (Sedlacek et al., “Flavopiridol (L86–8275; NSC 649890), A New Kinase Inhibitor for Tumor Therapy,” Int. J. Oncol., vol. 9 (1996), pp. 1143–1168). Analogs of flavopiridol are the subject of other publications, for example, U.S. Pat. No. 5,733,920 to Mansuri et al. (International Publication No. WO 97/16447) and International Publication Nos. WO 97/42949, and WO 98/17662. Results with purine-based derivatives are described in Schow et al., Bioorg. Med. Chem. Lett., vol. 7 (1997), pp. 2697–2702; Grant et al., Proc. Amer. Assoc. Cancer Res,. vol. 39 (1998), Abst. 1207; Legravend et al., Bioorg. Med. Chem. Lett., vol. 8 (1998), pp. 793–798; Gray et al., Science, vol. 281 (1998), pp. 533–538; Chang, et al., Chemistry & Biology, Vol. 6 (1999), pp. 361–375, WO 99/02162, WO 99/43675, and WO 99/43676. In addition, the following publications disclose certain pyrimidines that inhibit cyclin-dependent kinases and growth-factor mediated kinases: International Publication No. WO 98/33798; Ruetz et al., Proc. Amer. Assoc. Cancer Res,. vol. 39 (1998), Abst. 3796; and Meyer et al., Proc. Amer. Assoc. Cancer Res., vol. 39 (1998), Abst. 3794.
Benzensulfonamides that block cells in G1 are in development by Eisai, see Owa, et al., J. Med. Chem., Vol. 42 (1999), pp. 3789–3799. An oxindole CDK inhibitor is in development by Glaxo-Wellcome, see Luzzio, et al., Proc. Amer. Assoc. Cancer Res., Vol. (1999), Abst. 4102 and WO99/15500. Paullones were found in collaboration with the NCI, Schultz, et al., J. Med. Chem., Vol. (1999), pp. 2909–2919. Indenopyrazoles are described in WO99/17769 and by Seitz, et al, 218th ACS Natl. Mtg. (Aug. 22–26, 1999, New Orleans), Abst MEDI 316. Aminothiazoles are used in WO99/24416 and WO99/21845.
CHK1 is another protein kinase. CHK 1 plays an important role as a checkpoint in cell cycle progression. Checkpoints are control systems that coordinate cell cycle progression by influencing the formation, activation and subsequent inactivation of the cyclin-dependent kinases. Checkpoints prevent cell cycle progression at inappropriate times, maintain the metabolic balance of cells while the cell is arrested, and in some instances can induce apoptosis (programmed cell death) when the requirements of the checkpoint have not been met. See, e.g., O'Connor, Cancer Surveys, 29, 151–182 (1997); Nurse, Cell, 91, 865–867 (1997); Hartwell et al., Science, 266, 1821–1828 (1994); Hartwell et al., Science, 246, 629–634 (1989).
One series of checkpoints monitors the integrity of the genome and, upon sensing DNA damage, these “DNA damage checkpoints” block cell cycle progression in G1 & G2 phases, and slow progression through S phase. O'Connor, Cancer Surveys, 29, 151–182 (1997); Hartwell et al., Science, 266, 1821–1828 (1994). This action enables DNA repair processes to complete their tasks before replication of the genome and subsequent separation of this genetic material into new daughter cells takes place. Importantly, the most commonly mutated gene in human cancer, the p53 tumor suppressor gene, produces a DNA damage checkpoint protein that blocks cell cycle progression in G1 phase and/or induces apoptosis (programmed cell death) following DNA damage. Hartwell et al., Science, 266, 1821–1828 (1994). The p53 tumor suppressor has also been shown to strengthen the action of a DNA damage checkpoint in G2 phase of the cell cycle. See, e.g., Bunz et al., Science, 28, 1497–1501 (1998); Winters et al., Oncogene, 17, 673–684 (1998); Thompson, Oncogene, 15, 3025–3035 (1997).
Given the pivotal nature of the p53 tumor suppressor pathway in human cancer, therapeutic interventions that exploit vulnerabilities in p53-defective cancer have been actively sought. One emerging vulnerability lies in the operation of the G2 checkpoint in p53 defective cancer cells. Cancer cells, because they lack G1 checkpoint control, are particularly vulnerable to abrogation of the last remaining barrier protecting them from the cancer killing effects of DNA-damaging agents: the G2 checkpoint. The G2 checkpoint is regulated by a control system that has been conserved from yeast to humans. Important in this conserved system is a kinase, CHK1, which transduces signals from the DNA-damage sensory complex to inhibit activation of the cyclin B/Cdc2 kinase, which promotes mitotic entry. See, e.g., Peng et al., Science, 277, 1501–1505 (1997); Sanchez et al., Science, 277, 1497–1501 (1997). Inactivation of CHK1 has been shown to both abrogate G2 arrest induced by DNA damage inflicted by either anticancer agents or endogenous DNA damage, as well as result in preferential killing of the resulting checkpoint defective cells. See, e.g., Nurse, Cell, 91, 865–867 (1997); Weinert, Science, 277, 1450–1451 (1997); Walworth et al., Nature, 363, 368–371 (1993); and Al-Khodairy et al., Molec. Biol. Cell, 5, 147–160 (1994).
Selective manipulation of checkpoint control in cancer cells could afford broad utilization in cancer chemotherapeutic and radiotherapy regimens and may, in addition, offer a common hallmark of human cancer “genomic instability” to be exploited as the selective basis for the destruction of cancer cells. A number of factors place CHK1 as a pivotal target in DNA-damage checkpoint control. The elucidation of inhibitors of this and functionally related kinases such as CDS1/CHK2, a kinase recently discovered to cooperate with CHK1 in regulating S phase progression (see Zeng et al., Nature, 395, 507–510 (1998); Matsuoka, Science, 282, 1893–1897 (1998)), could provide valuable new therapeutic entities for the treatment of cancer.
Another group of kinases are the tyrosine kinases. Tyrosine kinases can be of the receptor type (having extracellular, transmembrane and intracellular domains) or the non-receptor type (being wholly intracellular). At least one of the non-receptor protein tyrosine kinases, namely, LCK, is believed to mediate the transduction in T-cells of a signal from the interaction of a cell-surface protein (Cd4) with a cross-linked anti-Cd4 antibody. A more detailed discussion of non-receptor tyrosine kinases is provided in Bolen, Oncogene, 8, 2025–2031 (1993), which is incorporated herein by reference.
In addition to its role in cell-cycle control, protein kinases also play a crucial role in angiogenesis, which is the mechanism by which new capillaries are formed from existing vessels. When required, the vascular system has the potential to generate new capillary networks in order to maintain the proper functioning of tissues and organs. In the adult, however, angiogenesis is fairly limited, occurring only in the process of wound healing and neovascularization of the endometrium during menstruation. See Merenmies, J., Parada, L. F., Henkemeyer, M., Cell Growth & Differentiation, 8, 3–10 (1997). On the other hand, unwanted angiogenesis is a hallmark of several diseases, such as retinopathies, psoriasis, rheumatoid arthritis, age-related macular degeneneration, and cancer (solid tumors). Folkman, Nature Med., 1, 27–31 (1995). Protein kinases which have been shown to be involved in the angiogenic process include three members of the growth factor receptor tyrosine kinase family: VEGF-R2 (vascular endothelial growth factor receptor 2, also know as KDR (kinase insert domain receptor) and as FLK-1); FGF-R (fibroblast growth factor receptor); and TEK (also known as Tie-2).
VEGF-R2, which is expressed only on endothelial cells, binds the potent angiogenic growth factor VEGF and mediates the subsequent signal transduction through activation of its intracellular kinase activity. Thus, it is expected that direct inhibition of the kinase activity of VEGF-R2 will result in the reduction of angiogenesis even in the presence of exogenous VEGF (see Strawn et al., Cancer Research, 56, 3540–3545 (1996)), as has been shown with mutants of VEGF-R2 which fail to mediate signal transduction. Millauer et al., Cancer Research, 56, 1615–1620 (1996). Furthermore, VEGF-R2 appears to have no function in the adult beyond that of mediating the angiogenic activity of VEGF. Therefore, a selective inhibitor of the kinase activity of VEGF-R2 would be expected to exhibit little toxicity.
Similarly, FGF-R binds the angiogenic growth factors aFGF and bFGF and mediates subsequent intracellular signal transduction. Recently, it has been suggested that growth factors such as bFGF may play a critical role in inducing angiogenesis in solid tumors that have reached a certain size. Yoshiji et al., Cancer Research, 57, 3924–3928 (1997). Unlike VEGF-R2, however, FGF-R is expressed in a number of different cell types throughout the body and may or may not play important roles in other normal physiological processes in the adult. Nonetheless, systemic administration of a small molecule inhibitor of the kinase activity of FGF-R has been reported to block bFGF-induced angiogenesis in mice without apparent toxicity. Mohammad et al., EMBO Journal, 17, 5996–5904 (1998).
TEK (also known as Tie-2) is another receptor tyrosine kinase expressed only on endothelial cells which has been shown to play a role in angiogenesis. The binding of the factor angiopoietin-1 results in autophosphorylation of the kinase domain of TEK and results in a signal transduction process which appears to mediate the interaction of endothelial cells with peri-endothelial support cells, thereby facilitating the maturation of newly formed blood vessels. The factor angiopoietin-2, on the other hand, appears to antagonize the action of angiopoietin-1 on TEK and disrupts angiogenesis. Maisonpierre et al., Science, 277, 55–60 (1997).
As a result of the above-described developments, it has been proposed to treat angiogenesis by the use of compounds inhibiting the kinase activity of VEGF-R2, FGF-R, and/or TEK. For example, WIPO International Publication No. WO 97/34876 discloses certain cinnoline derivatives that are inhibitors of VEGF-R2, which may be used for the treatment of disease states associated with abnormal angiogenesis and/or increased vascular permeability such as cancer, diabetes, psoriosis, rheumatoid arthritis, Kaposi's sarcoma, haemangioma, acute and chronic nephropathies, atheroma, arterial restinosis, autoimmune diseases, acute inflammation and ocular diseases with retinal vessel proliferation.
In addition to the protein kinases identified above, many other protein kinases have been considered to be therapeutic targets, and numerous publications disclose inhibitors of kinase activity, as reviewed in the following: McMahon et al., Current Opinion in Drug Discovery & Development, 1, 131–146 (1998); Strawn et al., Exp. Opin. Invest. Drugs, 7, 553–573 (1998).
There is still a need, however, for other small-molecule compounds that may be readily synthesized and are potent inhibitors of cell proliferation, for example, inhibitors of one or more protein kinases, such as CHK1, VEGF, CDKs or CDK/cyclin complexes. Because CDK4 may serve as a general activator of cell division in most cells, and because complexes of CDK4/cyclin D and CDK2/cyclin E govern the early G1 phase of the cell cycle, there is a need for effective and specific inhibitors of CDK4 and/or CDK2 for treating one or more types of tumors.